The long term objective of this project is to understand the physiological functions and regulation of protein tyrosine phosphatases (PTPs). Tyrosine phosphorylation plays pivotal roles in regulating cell proliferation, differentiation, and oncogenic transformation. The importance of these regulatory mechanisms, such as the activation of the mitogen activated protein (MAP) kinase, is shown to be conserved from yeast to human. To understand the physiological function and regulation of PTPs, the budding yeast Saccharomyces cerevisiae is chosen as the model system because both mechanism and molecular genetic methods can be readily applied in this organism. Preliminary studies from our laboratory demonstrate that PTP2 and PTP3 have overlapping functions in the mating pheromone signal transduction and are required for meiosis and sporulation. PTP2/PTP3 may be involved in the recovery of mating by directly inactivating the pheromone responsive FUS3/KSS1 MAP kinases. The first aim is to understand the molecular mechanisms of PTPs in regulation of mating pheromone signal transduction. The physiological functions of PTP2/PTP3 in regulation of the FUS3/KSS1 MAP kinases will be elucidated. Both genetic and biochemical experiments will be performed to demonstrate that FUS3/KSS1 are the direct in vivo substrates of PTP2/PTP3. Functional relationship among PTPs will be tested by combinatorial disruptions. New regulators of the pheromone activated MAP kinase pathway will be isolated and characterized. The second specific aim is to study the physiological functions and regulation of PTP2 and PTP3 in meiosis and sporulation. The requirements of PTP2/PTP3 for meiosis/sporulation demonstrate that tyrosine phosphorylation plays an essential role in the regulation of this process. The role of PTP2/PTP3 in meiotic initiation and commitment will be determined. The significance of PTP2/PTP3 for either ploidy or nutritional control of sporulation will be elucidated. In vivo, substrates whose dephosphorylation is essential for sporulation will be identified and characterized using both biochemical and molecular genetic approaches. Experiments in this proposal aim to establish the relationships between phosphatases, their substrates, and the biological functions they carry out by focusing on the molecular mechanisms of PTP2 and PTP3 in regulation of the mating pheromone signal transduction and the meiosis/sporulation processes. The completion of this proposal will provide important information of PTP in cellular regulation and build a valuable model for the functions and regulation of PTP in higher eukaryotes.